Antinuclear Antibody Indirect Immunofluorescence Research Articles

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America

Introduction/ObjectiveThis study aimed to describe the frequency of antinuclear antibody (ANA) staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America. Materials and methodsThis retrospective, descriptive, and observational study evaluated data from all patients undergoing antinuclear antibody indirect immunofluorescence assay from a single-tertiary center (University Hospital Fundación Valle del Lili, Cali-Colombia) in 2020. ResultsOne thousand and eight patients met the inclusion criteria. The median patient age was 47 (34–59.2) years, and most were female (769, 75.3%). A positive ANA immunofluorescence assay was observed in approximately two-thirds of patients (664, 65.8%). ANA test results were primarily used to exclude a suspected diagnosis in approximately half of the patients (466, 46.2%). Thirty-seven percent (250/664) of the cohort with ANA-positive titers had a systemic autoimmune rheumatic disease (SARD). The most prevalent SARDs included rheumatoid arthritis (RA) (55, 8.2%) followed by systemic lupus erythematosus (SLE) (37, 5.5%). The vast majority of ANA-positive patients had a reported speckled pattern (anti-cell [AC]-2,4,5; 269; 40.5%) followed by homogenous (AC-1; 266; 40%), nucleolar (AC-8,9,10; 46; 6.9%), and centromere (AC-3; 16; 2.4%). The most frequent patterns observed among SLE patients included homogenous (AC-1) patterns in 17 (45.9%) patients, speckled (AC-2,4,5) nuclear patterns in 11 (29.7%) patients, mixed patterns in 7 (18.9%) patients, and reticular/anti-mitochondrial antibody (AMA, AC-21) cytoplasmic patterns in 2 (5.4%) patients. ConclusionThis study is the first to describe ANA patterns in a Colombian population. Speckled and homogenous patterns were predominant in patients with SARDs.

Retrospective evaluation of "Rods and Rings" pattern detected in the anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) test.

Anti-rods and rings (anti-RR) antibodies have recently been described as a cytoplasmic pattern in IIF-based screening of autoantibodies on HEp-2 cells and ICAP has named it as AC-23. It is most frequently related to drug-induced antibody generation. This study aimed to investigate the clinical significance of AC-23 positivity and its relevance to the diagnosis and/or follow-up of the associated diseases and/or drug use. A multicenter retrospective study was conducted among 10 hospitals from six different provinces in Türkiye from January 2017 to December 2021. The laboratory data and clinical information of 600 patients with positive anti-RR antibodies out of 547.558 HEp-2 IIF ANA samples were analyzed. The distribution of AC-23 positive patients by year indicated a steady increase between 2017-2021. Anti-RR prevalence in post-COVID-19 period was significantly higher than that of pre-COVID-19 period (p=0.00). Concomitant ANA positivity was detected in 56.5% of patients, the most common patterns being AC-4 and AC-5 (41.1%). The most frequent pathology among the anti-RR positive patients was an autoimmune disease (19.83%); 28.57% of which had rheumatoid arthritis and 17.65% autoimmune liver disease. Among the 600 patients, 65 (10.83%) were diagnosed as hepatitis C virus (HCV) infection. Available data for 38 of the HCV patients revealed that 71.05% of them had a history of interferon alfa+ribavirin and 28.95% of them had a history of NS3/4/5A/5B polymerase inhibitor or protease inhibitor drug use. Significant increase in the rate of anti-RR positivity was observed in the post-COVID-19 period when compared to pre-COVID-19 period (p:0.00). This is the first multicenter study in Türkiye about the clinical association of anti-RR antibodies which may be ignored during routine HEp-2 IIF testing. Pathologies other than HCV should be taken into consideration in terms of the possible role of anti-RR in autoimmune diseases and other pathologies. The preliminary data obtained in this study suggest that anti-RR antibody development might also be associated to COVID-19, supporting the several previous data related to the potential of viruses triggering the formation of autoantibodies. Large-scale prospective studies should elucidate the clinical significance of RR pattern and determine its role in patient diagnosis and follow-up.

Retrospective analysis of the clinical significance of Ro52/TRIM21 antibody and specific antinuclear antibody patterns by indirect immunofluorescence.

To determine the clinical significance of Ro52 protein/tripartite motif-containing 21 antibody and specific antinuclear antibody patterns using indirect immunofluorescence technique. The retrospective study was conducted at the clinical laboratory of the First Affiliated Hospital of Chongqing Medical University, China, and comprised data from January 2017 to December 2021 of patients who underwent antinuclear antibody and anti-extractable nuclear antigen antibody detection. Inpatients with Ro52 antibody-positive status were taken as the cases, while anti-Ro52 negative patients with clear clinical diagnosis were taken as the controls. Data was analysed using SPSS 19. There were 1802 cases and 1211 controls. Positive Ro52 showed significantly greater frequency in patients with primary Sjogren's syndrome, systemic lupus erythematosus, inflammatory myositis, dry eyes and interstitial lung disease (p<0.05). Ro52 antibody showed high positive predictive value for primary Sjogren's syndrome 25(96.15%), systemic lupus erythematosus 259(91.20%), connective tissue disease-associated interstitial lung disease 45(86.67%) and inflammatory myositis 60(86.67%). Antinuclear antibody indirect immunofluorescence patterns most frequently detected were nuclear speckled 128(40.89%) and cytoplasmic speckled 126(40.26%) (p<0.05). Interstitial lung disease was associated with the presence of cytoplasmic speckled antinuclear antibody indirect immunofluorescence pattern 24(19.2%), while tumours 47(36.5%) and hepatitis B 26(20.3%) seemed to be more frequent with nuclear speckled pattern (p<0.05). The simultaneous reactivity extractable nuclear antigen antibodies most frequently detected were antinuclear antibody+Ro52+anti-Sjogren's syndrome A+ 558(33.96%). Ro52 antibody positivity was found to be associated with Sjogren's syndrome, systemic lupus erythematosus, inflammatory myositis, dry eye and interstitial lung disease. The antinuclear antibody immunofluorescence pattern of Ro52 positive was single and primarily granular cytoplasm type. Antinuclear antibody negative and Ro52 positive in the serum of patients also had certain significance in auxiliary disease diagnosis.

Diagnostic Validation of Fully Automated ANA Detection by Indirect Immunofluorescence: An Initiative towards Standardization

Objective: This study entailed the diagnostic validation of an automated system utilized for the detection of antinuclear antibodies (ANA) through the indirect immunofluorescence method, juxtaposed with the traditional manual approach for ANA detection. The study was undertaken in the Department of Chemical Pathology at Chughtai Laboratory, Lahore, spanning a period of three months, from December 2018 to February 2019. Methods: A total of 132 patient samples were gathered for the purpose of conducting this validation study. The manual detection of ANA was performed by using slides coated with HEp-2 cells. An LED fluorescence microscope was used for the interpretation of results by a trained technologist as well as by a pathologist. After manual detection of ANA, the same specimens were evaluated by a fully automated ANA-IIF detection system which has inbuilt characteristics of slide processing, microscopy, image capturing and displaying specific ANA patterns. The results were interpreted as positive for those samples having sample titers more than 1:80. The same criteria of positivity were used for both manual and automatic systems. Results: A total of 132 specimens underwent evaluation through both manual and automated ANA indirect immunofluorescence (IIF) detection systems. The concordance between these two methodologies exhibited an overall agreement of 95.7%, with a positive agreement of 97.5% and a negative agreement of 94.9%. Cohen's kappa value stood at 91.7% (ranging from 83.4% to 98.9%), indicating a robust correlation between the two detection systems. The diagnostic sensitivity of the fully automated IIF methodology demonstrated 89.38% (with a 95% confidence interval of 77.24% to 96.13%) and the diagnostic specificity of 95.22% (with a 95% confidence interval of 87.88% to 98.76%). Conclusion: The fully automated showed strong correlation with manual ANA detection methodology based on the principle of indirect immunofluorescence.

The Discrepancy of ANA and Compartment Bead Patterns Suggestive of a Neuropsychiatry Systemic Lupus Erythematosus (NPSLE)

Neuropsychiatric systemic lupus erythematosus (NPSLE) exhibits neurological and psychiatric manifestations in systemic lupus erythematosus (SLE) patients, which NPSLE diagnosis can be challenging for rheumatologists. An Indonesian female, 44 years old, complained of two times seizures with 10-min duration, which during seizures were stiff, eyes rolled up, foaming at the mouth, wet the bed, and fainting afterward. The patient also has a history of SLE and received cyclophosphamide therapy 5 years ago. Her clinical condition showed facial and lingual palsy, with central type on the right. Antinuclear antibody indirect immunofluorescence (ANA IF) positive using cytobead ANA with a homogenous pattern and cytoplasmic speckled titer 1/80. Confirmation beads showed positive of dsDNA only. ANA profile showed positive antinucleosome, antihistone, and AMA-M2, and also increased anticardiolipin antibody that supports the diagnosis of NPSLE. The difference in the pattern of ANA IF with confirmation beads suggests the presence of other autoantibodies in NPSLE.

Effect of COVID-19 on ANA positivity in the Indian population

The prevalence of Antinuclear Antibodies (ANA) positivity has increased following Covid-19 infection. This study investigates the ANA positivity rate by comparing ANA data from two distinct years, 2019 (a pre-Covid year) and 2022 (a post-Covid year). This retrospective study analyzes and compares ANA Indirect Immunofluorescence Assay (IFA) data for the years 2019 and 2022 across various parameters, including age, gender, prevalence rate, positivity rate by grade, and patterns of ANA. In the post-Covid year 2022, there was a notable increase in both the total suspected cases and the ANA-positive cases, amounting to approximately a 30% rise. Positivity rates were observed to increase with age, and a female preponderance was noted in both years. Nuclear speckled patterns remained the most common in both time periods. The post-Covid pandemic period has witnessed a significant role of immune modulation in the development of autoimmunity. This phenomenon could potentially be attributed to Molecular Mimicry, the production of Autoantibodies upon exposure to Viral epitopes through the generation of Neutrophil Extracellular Traps (NETs), or via Toll-like Receptor (TLR) pathways of immune modulation, which may activate latent autoimmunity.

B-073 The Clinical Utility of Reporting AC-2 Pattern With Anti-DFS70 Antibody

Abstract Background The dense fine speckled (DFS) pattern (AC-2) is often observed in antinuclear antibody (ANA) indirect immunofluorescence (IIF) assay, and the related anti-DFS70 antibody is known to be negatively associated with a systemic autoimmune rheumatic disease (SARD). Identifying the AC-2 pattern is challenging, as it could be generally confused with the AC-1, AC-4, AC-5, or AC-29 patterns displaying stained metaphase plates or speckled nuclei. Since the anti-DFS70 antibody is not included in routine autoantibody tests, the clinical utility of the AC-2 pattern and anti-DFS70 antibody is not adequately applied in the laboratory and clinical practice. We evaluated the prevalence of anti-DFS70 and other autoantibodies in samples presenting AC-2 pattern in ANA IIF assay. Additionally, we analyzed the association of anti-DFS70 antibodies and clinical features in patients with AC-2 patterns. Method We retrospectively reviewed the ANA IIF images of 1839 samples made by an automated ANA analyzer classified as AC-1, AC-2, AC-4, AC-5, and AC-29 patterns. AC-1, AC-2, AC-4, AC-5, and AC-29 patterns comprised 60.5% of all ANA positives (1839/3039). Among them, the AC-2 pattern was identified in 18.5% (341/1839) by two experts, and only 197 with residual samples were available for further evaluation. Those 197 samples were tested for anti-DFS70 antibodies with enzyme-linked immunosorbent assay (ELISA) and line immunoblot assay (LIA). They were also tested for 22 other autoantibodies (dsDNA, nucleosomes, histones, SS-A/Ro-52, SS-B, RNP/Sm, Sm, Mi-2α, Mi-2β, Ku, CENP-A, CENP-B, Sp100, PML, Scl-70, PM/Scl100, PM/Scl75, RP11, RP155, gp210, PCNA) with LIA. Clinical information from 197 patients was obtained from a retrospective review of electronic medical records. Results Anti-DFS70 antibody prevalence by ELISA and LIA was 32.0% (63/197) and 19.8% (39/197), respectively. The agreement percentage between the two assays was 81.7%, with a kappa value of 0.53 (95% confidence interval: 0.40–0.66). Of the 69 DFS70+ samples (as per ELISA or LIA), the coexistence of other autoantibodies was found in 81.2% (56/69). Of the 128 DFS70- samples, 90.6% (116/128) revealed other autoantibodies. The three most frequent autoantibodies (except anti-DFS70) in 197 patients with AC-2 patterns were anti-SS-A/Ro-52 (30.5%, 60/197), anti-nucleosomes (27.4%, 54/197), and anti-histones (20.3%, 40/197). There was no significant difference in the prevalence of the other 22 autoantibodies between DFS70+ and DFS70− patients. The anti-DFS70 prevalence in the SARD group was 33.3% (50/150), and in the non-SARD was 40.4% (19/47) without significant difference. However, of the 69 DFS70+ patients, the isolated anti-DFS70 was found more frequently in the non-SARD group (36.8%, 7/19) than in the SARD group (12.0%, 6/50) (P = 0.0346). Conclusion The anti-DFS70 positive rate in AC-2 pattern samples is lower than a third, and the prevalence is affected by different detection assays. Along with the AC-2 pattern, just reporting the presence of anti-DFS70 is not valuable for SARD exclusion. Therefore, it is important to co-examine other autoantibodies, such as anti-SS-A, anti-nucleosomes, and anti-histones, to confirm the isolated anti-DFS70.

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America

Introduction/ObjectiveThis study aimed to describe the frequency of antinuclear antibody (ANA) staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America. Materials and methodsThis retrospective, descriptive, and observational study evaluated data from all patients undergoing antinuclear antibody indirect immunofluorescence assay from a single-tertiary center (University Hospital Fundación Valle del Lili, Cali-Colombia) in 2020. ResultsOne thousand and eight patients met the inclusion criteria. The median patient age was 47 (34–59.2) years, and most were female (769, 75.3%). A positive ANA immunofluorescence assay was observed in approximately two-thirds of patients (664, 65.8%). ANA test results were primarily used to exclude a suspected diagnosis in approximately half of the patients (466, 46.2%). Thirty-seven percent (250/664) of the cohort with ANA-positive titers had a systemic autoimmune rheumatic disease (SARD). The most prevalent SARDs included rheumatoid arthritis (RA) (55, 8.2%) followed by systemic lupus erythematosus (SLE) (37, 5.5%). The vast majority of ANA-positive patients had a reported speckled pattern (anti-cell [AC]-2,4,5; 269; 40.5%) followed by homogenous (AC-1; 266; 40%), nucleolar (AC-8,9,10; 46; 6.9%), and centromere (AC-3; 16; 2.4%). The most frequent patterns observed among SLE patients included homogenous (AC-1) patterns in 17 (45.9%) patients, speckled (AC-2,4,5) nuclear patterns in 11 (29.7%) patients, mixed patterns in 7 (18.9%) patients, and reticular/anti-mitochondrial antibody (AMA, AC-21) cytoplasmic patterns in 2 (5.4%) patients. ConclusionThis study is the first to describe ANA patterns in a Colombian population. Speckled and homogenous patterns were predominant in patients with SARDs.

Negative ANA-IIF in SLE patients: what is beyond?

The antinuclear antibody (ANA) test has high sensitivity in diagnosing and classifying systemic lupus erythematosus (SLE).ObjectivesTo describe the immunological pattern of SLE patients through investigating specific antinuclear autoantibodies by enzyme dot immunoassay and studying their frequency in both positive and negative ANA indirect immunofluorescence assay (IIF) cases.MethodsIn a cross-sectional study, blood samples from 393 newly diagnosed SLE patients were analyzed using (IIF) on HEp-2 cells and ANA dot immunoassay by automated enzyme immunoassay (EIA) to detect 19 antibodies.ResultsNinety-one percent of the patients are females; their mean age was 37 ± 12.28. Antinuclear antibody (ANA) was detected by IIF in 82.4% of cases, with 181 (46.1%) speckled and 167 (42.4%) homogeneous ANA patterns. The majority of patients (96%) demonstrated autoantibodies via EIA. Among the ANA-IIF-negative patients, 97.2% demonstrated autoantibodies. There was a significant difference in the frequency of certain autoantibodies between SLE patients with negative and positive ANA-IIF (1.44 0.73, 3.12 2.09, p = 0.00) respectively.ConclusionThe results of analyzing 19 autoantibodies with the ANA staining pattern increased the significance of analyzing the immune profile even if IIF is negative when clinical symptoms strongly suggest SLE diagnosis. Certain autoantibodies may evade staining by the IFA approach while they are present in the patient’s serum, and they may not be detected by the ANA EIA profile if it does not contain that antigenic substrate.Key Points• Indirect immunofluorescence on Hep-2 is the conventional method for ANA detection and is regarded as the “gold standard” for testing in clinical practice for SLE.• In our study, ANA profile dot enzyme immunoassay (EIA)-based test was performed to evaluate 19 autoantibodies in SLE patients either positive or negative for ANA-IIF.• The presence of anti-dsDNA with ANA-IIF-negative serum in 32.4% of SLE patients provides evidence that not all anti-dsDNA antibodies are identified on standard HEp-2 substrates.• certain autoantibodies can evade staining by the ANA-IIF method despite being present in the SLE patient’s blood; this supports the ANA profile enzyme dot immunoassay as a more sensitive test.

КЛИНИКО-ДИАГНОСТИЧЕСКОЕ ЗНАЧЕНИЕ АНТИНУКЛЕАРНЫХ АНТИТЕЛ В ПРАКТИКЕ ПЕДИАТРА (ЧАСТЬ I)

In recent decades, there has been an increase in the incidence of various autoimmune, including rheumatic diseases, while their debut has been shifted to an earlier age. The clinical picture of rheumatic diseases is distinguished by severe polymorphism; therefore, laboratory diagnostic methods are important for diagnostic search. One of the most widely used tests in the rheumatological practice is the detection and identification of antinuclear antibodies (ANA). ANA are a large group of autoantibodies (auto-Abs) directed against various cell structures, including not only the nucleus but also the nuclear membrane, mitotic apparatus, cytoplasm components and cell organelles, as well as cell membranes. Different methods are used for testing, but the method of ANA detection through indirect immunofluorescence antinuclear antibody test (IF-ANA) is recommended as a “gold standard” with human laryngeal carcinoma HEp-2 cells being recognized as a standard substrate. IF-ANA with HEp-2 has the highest diagnostic sensitivity compared with other methods, it includes the phase of serial dilutions of positive serums and a visual analysis of the type of glow, the list of which currently has 30 patterns. ANA glow types are caused by various auto-Abs that respond with antigens located in the nucleus and cytoplasm of the HEp-2 cells line. Assessment of the type of glow in conjunction with clinical and laboratory data allows planning of further examination with confirming tests to determine the specificity of ANA, some of which, in particular, are auto-Abs to DFS70 antigen, which in the absence of other types of autoantibodies, are more likely to doubt the presence of a systemic rheumatic disease. The diagnostic specificity of various screening techniques for ANA detection is relatively low, they are detected with a fairly high frequency in patients with other diseases and in healthy individuals of different gender and age, including children.

Sex-Dependent Differences in Serum Autoantibody Levels in the 3×Tg-AD Model of Alzheimer’s Disease

Abstract Sex-dependent discrepancies in disease prevalence and serum autoantibody levels are observed in patients and animal models of Alzheimer’s disease (AD). The present study examines whether gonadal hormones play a role in sex differences in serum autoantibody levels in the 3×Tg-AD mouse model of AD. 3×Tg-AD and wild-type (WT) mice were gonadectomised or sham-operated at 3 months of age. After behavioural phenotyping at 6 months of age, the animals were assessed for serum autoantibodies by indirect immunofluorescence for antinuclear antibodies (ANA) and by line-immunoblot assay for an additional 16 monospecific autoantibodies including anti-nucleosome antibodies. There were significant differences between the strains in ANA levels, with the major target antigens confirmed as nucleosomes. The results of ANA and anti-nucleosome assays were combined for further analysis. Further analysis revealed: 1) the level of serum autoantibodies in male 3×Tg-AD mice was higher than in female 3×Tg-AD animals, and this was not altered by orchiectomy. 2) sham-operated 3×Tg-AD female mice displayed a significantly lower level of serum autoantibodies than sham-operated WT females. 3) ovariectomy further reduced the level of serum autoantibodies in female 3×Tg-AD mice. The results suggest that dissimilar levels of serum autoantibodies in 3xTg-AD mice are a sex-dependent phenomenon and that female hormones play a role in regulation of their synthesis. Funded by grant #SVB-158618 from the Canadian Institutes of Health Research to MF.

Anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase antibodies: incidence using different testing criteria, and case series.

To estimate the incidence of anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies utilising different testing criteria, and review the clinical details of a series of patients with associated autoimmune myopathy. The incidence of anti-HMGCR antibodies in 2019 from 3 groups, South West London, Berkshire/Surrey and Southampton, were compared in the adult population. Anti-HMGCR antibodies were measured by commercial chemiluminescent and immunodot assays. The case notes of patients with anti-HMGCR antibodies were reviewed for the case series. The estimated incidence of anti-HMGCR antibodies in the first 2 groups was 1.94 per million adults per year, and in the third group 10.3 per million adults per year. In the first 2 groups the test criteria restricted analysis to specific clinician request for anti-HMGCR. In the third group test criteria included cases with less specific clinical features or a cytoplasmic indirect immunofluorescence anti-nuclear antibody pattern. The latter strategy had a positive predictive value of 66.1% for anti-HMGCR associated myopathy. A case series of 27 patients with anti-HMGCR antibodies revealed 19 with myopathy, oesophageal involvement in 26% and median peak CK 8000 IU/L. Response to treatment, including intravenous immunoglobulin, was good with CK normalising after median 5.5 months. In 8 cases there was no evidence of autoimmune muscle disease, 7 not statin exposed. Varying criteria result in a 5-fold difference in estimated incidence of anti-HMGCR antibodies, revealing positive cases without evidence of myopathy. Patients with anti-HMGCR myopathy respond well to immune suppression, supporting wider testing for these antibodies amongst patients with myopathy.

Determination and Prevalence of Antinuclear Antibody (ANA) Patterns in Autoimmune Disorders in a Tertiary Care Hospital, Jaipur

Introduction: Autoimmunity is the immune response of antibodies against normal cellular components. With the advent of serological testing for autoimmune disorders, detection of anti-nuclear antibodies (ANA) by indirect immunofluorescence method became the gold standard. ANA screening is non-specific, reliable and a quick way and identifies immunofluorescent patterns linked to autoimmune disorders. Aim: This study was undertaken to identify the prevalence of ANA patterns amongst the various demo graph presented in a tertiary care hospital in Jaipur, Rajasthan. Material and Method: 100 serum samples were screened by indirect immunofluorescence microscopy in the duration of July 2021 to October 2021. Results: 27% of the serum samples presented with positive ANA patterns out of which 62.96% were nuclear speckled, followed by 22.22% of nuclear homogenous pattern and 7.40% were observed to be cytoplasmic speckled. The age bracket with higher positivity were 20-40 years (28%) and 40-60 (28%). There was female predominance noted in the ANA positivity. Conclusion: Nuclear speckled was the most frequent pattern, few unusual patterns were also observed. An observational study is needed to understand not only the epidemiology of autoimmune disorders but also the predictive value of ANA IIF in clinical setup, besides considering the possibility of taking up IIF as a screening tool for autoimmune disorders. Keywords: Antinuclear antibody (ANA), indirect immunofluorescence (IIF), systemic lupus erythematosus (SLE), autoantibody.

Antinuclear antibody patterns by indirect immunofluorescence test: An experience from a rural tertiary health centre of Puducherry, South India

Anti-nuclear antibodies (ANA) are known to be associated with autoimmune diseases and most commonly with connective tissue diseases (CTD). Screening for ANA is the first investigation of choice in the diagnosis of autoimmune diseases and indirect immunofluorescence (IIF) test using Hep-2 cells has been considered the gold standard method. The prevalence of autoimmune diseases and hence the ANA positivity and ANA IIF patterns differ in various geographical regions. This study was aimed to estimate the ANA positivity rates among patients with suspected or confirmed auto immune diseases and to determine the common ANA IIF patterns in the positive samples.: This is a retrospective study including 235 patients with either clinical suspicion of, or suffering from autoimmune diseases attending a tertiary health care hospital in Puducherry, South India, between November 2018 and April 2020. ANA IIF test (Hep 20-10/ primate liver, Euroimmun, Germany) was performed with 1:100 dilutions of patient serum.The ANA positivity rate was 14.5% (34 out of 235 samples were positive). ANA was positive in 10 (13.3%) males and 24 (15%) females. The mean age of ANA positive patients were 43.4 years.The most common pattern was nucleus fine granular (n=8) followed by cytoplasm fine granular (n=7) and centromere (n=4).A significant ANA positivity rate was found in our study and a detailed clinical study has to be conducted to draw further conclusions.

Laboratory strategy for autoantibodies testing as a diagnostic marker of pulmonary fibrosis in systemic sclerosis: A preliminary study prior to cohort registry of systemic sclerosis in West Java Indonesia

Background: Interstitial lung disease (ILD) is a complication often found in patients with Systemic Sclerosis (SSc). This usually manifests as pulmonary fibrosis and is the leading cause of death in SSc patients. Antinuclear antibodies indirect immunofluorescence (ANA-IIF) test and specific autoantibodies testing could be employed as a diagnostic marker to detect pulmonary fibrosis, but the high cost of these tests often burdens patients in developing countries. This study aims to compare ANA-IIF and an alternative, possibly inexpensive method to detect pulmonary fibrosis in SSc. Methods: This is a preliminary study prior to the cohort systemic sclerosis registry conducted in Rheumatology Outpatient Clinic and Clinical Pathology Department at Dr. Hasan Sadikin General Hospital, Bandung, from January 1, 2018 to December 31, 2020. Two contrastive laboratory testing strategies were followed to confirm pulmonary fibrosis in 61 SSc patients. The first strategy was to perform antinuclear antibodies indirect immunofluorescence (ANA-IIF) test followed by specific autoantibodies detection [anti-topoisomerase-I (ATA), Anti-Th/To, SSc Profile, Immunoblot]. The alternative strategy included conducting the tests for SSc autoantibodies in the reverse order. Results: A total of 42 out of 61 (68.8%) subjects were diagnosed with pulmonary fibrosis by high-resolution computed tomography (HRCT) scan. The first strategy detected dominant specific pulmonary fibrosis autoantibodies in subjects with pulmonary fibrosis: homogeneous nucleolar pattern was dominant (n = 25, 59.52%), followed by nucleolar pattern (n = 17, 40.48%). Of all the subjects with a homogeneous nucleolar pattern, ATA was detected in 15 subjects (60%), the combination of ATA and Th/To in 9 subjects (36%), and Ro52 in one subject (4%). The second strategy yielded positive autoantibodies results in 29 out of 42 (69.05%) subjects, ATA in 20 subjects (47.61%), and Th/To in 9 patients (21.44%), but failed to detect any autoantibodies specific to SSc-ILD in 13 subjects (30.95%). Conclusion: The first strategy [ANA-IIF followed specific autoantibodies testing (ATA, Th/To, SSc profile and immunoblot)] is more likely to detect specific autoantibodies toward ILD in SSc patients.

Comparison of antinuclear antibody profiles obtained using line immunoassay and fluorescence enzyme immunoassay.

ObjectiveLIA-ANA-Profile-17S is a multiplex line immunoassay that simultaneously detects 17 antinuclear antibodies (ANAs) against extractable nuclear antigens (ENAs). We evaluated the utility of LIA-ANA-Profile-17S as a supplement to ANA indirect immunofluorescence (IIF) and EliA ENA (a fluorescence enzyme immunoassay) for diagnosis of ANA-associated rheumatic diseases.MethodsSera were collected from 245 patients referred for an ANA IIF test. LIA-ANA-Profile-17S results were compared with those of EliA ENA. The kappa coefficients, agreement rates, and diagnostic performance of these tests were assessed for systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SjS).ResultsWe observed almost perfect interassay agreement for antibodies against Ro52/Ro60, CENP-B, and Scl-70 (kappa = 0.91, 0.97, and 1.00, respectively); strong agreement for anti-SS-B/La antibody (kappa = 0.81); and relatively low agreement for other antibodies, including those against dsDNA, Sm, RNP, and Jo-1. For SLE diagnosis, LIA-ANA-Profile-17S showed lower sensitivity and similar specificity compared with EliA ENA. The sensitivity and specificity of these two assays were similar for SjS diagnosis.ConclusionsThe specificity of LIA-ANA-Profile-17S was enhanced when combined with ANA IIF and was comparable with that of EliA ENA. LIA-ANA-Profile-17S showed relatively good agreement with EliA ENA. In combination with ANA IIF, these assays showed enhanced diagnostic performance.

Integrating quality assurance in autoimmunity: the changing face of the automated ANA IIF test.

Currently available computer-aided diagnosis (CAD) systems for the detection of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) assay enable a standardized measurement of system-specific fluorescent intensity (FI) measures. We aimed to evaluate an internal quality control (iQC) program that controls the total ANA IIF process in routine practice. In addition to the kit iQC materials, supplemental quality indicators were integrated in a total quality assurance (QA) program: patient-derived iQC's samples (negative, 1/160 fine speckled and 1/160 homogeneous), median sample FI per run and percentage of ANA IIF positive samples per run. Analytical rejection criteria were based on the imprecision of the positivity index (PI) measure of the Zenit PRO system (Menarini). Clinical rejection criteria were based on changes in FI that correspond to a change in ANA IIF titer of≥2. To evaluate the QA program, different artificial errors were introduced during the ANA IIF process. After every run, quality indicators were evaluated and compared to the pre-set target values. Rescanning the ANA IIF slides five times, using an old conjugate and a needle obstruction resulted in analytically and even clinically relevant errors in ANA IIF results. All errors were correctly detected by the different defined quality indicators. Traditional Westgard rules, including analytically (and clinically) defined rejection limits were useful in monitoring quality indicators. The integration of a total process iQC program in CAD systems, based on the specific FI measurands and performance criteria of the system, adds value to QA.

Current laboratory and clinical practices in reporting and interpreting anti-nuclear antibody indirect immunofluorescence (ANA IIF) patterns: results of an international survey

BackgroundThe International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) has recently proposed nomenclature in order to harmonize ANA indirect immunofluorescence (IIF) pattern reporting. ICAP distinguishes competent-level from expert-level patterns. A survey was organized to evaluate reporting, familiarity, and considered clinical value of ANA IIF patterns.MethodsTwo surveys were distributed by European Autoimmunity Standardization Initiative (EASI) working groups, the International Consensus on ANA Patterns (ICAP) and UK NEQAS to laboratory professionals and clinicians.Results438 laboratory professionals and 248 clinicians from 67 countries responded. Except for dense fine speckled (DFS), the nuclear competent patterns were reported by > 85% of the laboratories. Except for rods and rings, the cytoplasmic competent patterns were reported by > 72% of laboratories.Cytoplasmic IIF staining was considered ANA positive by 55% of clinicians and 62% of laboratory professionals, with geographical and expertise-related differences.Quantification of fluorescence intensity was considered clinically relevant for nuclear patterns, but less so for cytoplasmic and mitotic patterns. Combining IIF with specific extractable nuclear antigens (ENA)/dsDNA antibody testing was considered most informative.Of the nuclear competent patterns, the centromere and homogeneous pattern obtained the highest scores for clinical relevance and the DFS pattern the lowest. Of the cytoplasmic patterns, the reticular/mitochondria-like pattern obtained the highest scores for clinical relevance and the polar/Golgi-like and rods and rings patterns the lowest.ConclusionThis survey confirms that the major nuclear and cytoplasmic ANA IIF patterns are considered clinically important. There is no unanimity on classifying DFS, rods and rings and polar/Golgi-like as a competent pattern and on reporting cytoplasmic patterns as ANA IIF positive.

A multi-centre study for standardization of antinuclear antibody indirect immunofluorescence screening with automated system

Introductionndirect immunofluorescence assay (IFA) using HEp-2 as substrate plays a consolidate role for the detection and measurement of ANA, which is currently considered as the reference method for detection. Manual operation is still very common in China, therefore, the need of standardization and automation for ANA-IFA detecting has been highlighted. ObjectiveThe current multi-center study is aimed to evaluate if HELIOS (AESKU Diagnostics, Wendelsheim, Germany) contributes to comparability of ANA screening results among different labs，and establish application specification of HELIOS for standardization of ANA detection. MethodsANA detection by manual IFA method and HELIOS on 230 clinical serum samples in eight laboratories. The performance to discriminate positive/negative screening results, endpoint titer estimation and pattern recognition were evaluated in HELIOS and manual visual. ResultsThe positive coincident rate for ANA detection by manual IFA ranges from 87.7% to 97.8%, the negative coincidence rate ranges from 68.8% to 100%, the correctly estimated titer evaluation were 80 to 171 cases, the correct pattern in 146 to 161 cases, respectively. The positive coincident rate of HELIOS for ANA detection ranges from 91.2% to 97.7%, the negative coincidence rate ranges from 96.5% to 100%, the correctly estimated titer evaluation were 145 to 157 cases, the correct pattern in 123 to 140 cases, respectively. ConclusionHELIOS could provide accurate diagnostic results, this include not only positive/negative results, but also endpoint titer, common patterns. The application of this system can help to promote standardization of ANA detection.

Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing

Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6–97.5% of patterns and 71.0–93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.